Neoplasia is a disease characterized by an abnormal cell growth known as a neoplasm. Neoplasms may manifest in the form of a leukemia or a tumor, and may be benign or malignant. Malignant neoplasms, in particular, can result in a serious disease state, which may threaten life. Significant research efforts and resources have been directed toward the elucidation of antineoplastic measures, including chemotherapeutic agents, which are effective in treating patients suffering from neoplasia. Effective antineoplastic agents include those which inhibit or control the rapid proliferation of cells associated with neoplasms, those which effect regression or remission of neoplasms, and those which generally prolong the survival of patients suffering from neoplasia. Successful treatment of malignant neoplasia, or cancer, requires elimination of all malignant cells, whether they are found at the primary site, or have extended to local/regional areas, or have metastasized to other regions of the body. The major therapies for treating neoplasia are surgery and radiotherapy (for local and regional neoplasms) and chemotherapy (for systemic sites) (Beers and Berkow (eds.), The Merck Manual of Diagnosis and Therapy, 17th ed. (Whitehouse Station, N.J.: Merck Research Laboratories, 1999) 973-74, 976, 986, 988, 991).
Despite the various methods for detecting, diagnosing, and treating cancers, the disease remains prevalent in all segments of society, and is often fatal. Clearly, alternative strategies for detection (including the development of markers that can identify neoplasias at an early stage) and treatment are needed to improve survival in cancer patients. In particular, a better understanding of tumor suppressors, and tumor-suppression pathways, would provide a basis from which novel detection, diagnostic, and treatment regimens may be developed.
The p53 tumor suppressor exerts anti-proliferative effects, including growth arrest, apoptosis, and cell senescence, in response to various types of stress (Levine, A. J., p53, the cellular gatekeeper for growth and division. Cell, 88:323-31, 1997; Giaccia and Kastan, The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev., 12:2973-83, 1998; Prives and Hall, The p53 pathway. J. Pathol., 187:112-26, 1999; Oren, M., Regulation of the p53 tumor suppressor protein. J. Biol. Chem., 274, 36031-034, 1999; Vogelstein et al., Surfing the p53 network. Nature, 408:307-10, 2000; Michael and Oren, The p53-Mdm2 module and the ubiquitin system. Semin. Cancer Biol., 13:49-58, 2003). p53 is the most commonly mutated gene in human cancers, with more than 50% of tumors displaying some alteration in p53 (Hollstein et al., Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res., 22:3551-55, 1994; Hollstein et al., New approaches to understanding p53 gene tumor mutation spectra. Mutat. Res., 431:199-209, 1999).
Wild-type p53 has been called the guardian of the genome, as it responds to DNA damage or checkpoint failure by either arresting the cell in the G1 phase for damage repair, or initiating an apoptotic pathway to eliminate the damaged cell entirely (Lane, D. P., Nature, 358:15-16, 1992; Levine, A. J., p53, the cellular gatekeeper for growth and division. Cell, 88:323-31, 1997). p53 is also critical for maintenance of genomic stability, aberrant ploidy, gene amplification, increased recombination, and centrosomal dysregulation—all of which have been observed in cells lacking functional p53 (Donehower et al., Nature, 356:215-21, 1992). These observations suggest that abrogation of p53 function is critical in tumorigenesis of cancer. Additionally, numerous studies indicate that inactivation of the p53 pathway is a pivotal event in tumorigenesis of all kinds of human cancers, including breast cancer (Vogelstein et al., Surfing the p53 network. Nature, 408:307-10, 2000). Accumulating evidence further indicates that, in cells that retain wild-type p53, other defects in the p53 pathway play an important role in tumorigenesis (Prives and Hall, The p53 pathway. J. Pathol., 187:112-26, 1999; Oren, M., Regulation of the p53 tumor suppressor protein. J. Biol. Chem., 274, 36031-034, 1999).
p53 is a short-lived protein, the activity of which is maintained at low levels in normal cells. The molecular function of p53 that is required for tumor suppression involves the ability of p53 to act as a transcriptional factor in regulating endogenous gene expression. A number of genes which are critically involved in either cell-growth arrest or apoptosis have been identified as p53 direct targets, including p21CIP1/WAF1, Mdm2 (murine double minute 2), GADD45, cyclin G, 14-3-3σ, Noxa, p53AIP1, and PUMA (Kastan et al., A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell, 71:587-97, 1992; el-Deiry et al., WAF1, a potential mediator of p53 tumor suppression. Cell, 75:817-825, 1993; Wu et al., The p53-mdm-2 autoregulatory feedback loop. Genes Dev., 7:1126-32, 1993; Barak et al., mdm2 expression is induced by wild type p53 activity. EMBO J., 12:461-68, 1993; Okamoto and Beach, Cyclin G is a transcriptional target of the p53 tumor suppressor protein. EMBO J., 13:4816-22, 1994; Oda et al., Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science, 288:1053-58, 2000a; Oda et al., p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell, 102:849-62, 2000b; Nakano and Vousden, PUMA, a novel proapoptotic gene, is induced by p53. Molecular Cell, 7:683-94, 2001; Yu et al., PUMA induces the rapid apoptosis of colorectal cancer cells. Molecular Cell, 7:673-82, 2001). Furthermore, tight regulation of p53 itself is essential for its effect on tumorigenesis and the maintenance of normal cell growth.
Numerous studies imply the existence of multiple pathways involved in p53 stabilization (Shieh et al., DNA damage-induced phosphorylation of p53 alleviates inhibition MDM2. Cell, 91:325-34, 1997; Ashcroft et al., Regulation of p53 function and stability by phosphorylation. Mol. Cell Biol., 19:1751-58, 1999; Blattner et al., DNA damage induced p53 stabilization: no indication for an involvement of p53 phosphorylation. Oncogene, 18:1723-32, 1999; Dumaz and Meek, Serine15 phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM2. EMBO J., 18:7002-10, 1999; Ashcroft et al., Stress signals utilize multiple pathways to stabilize p53. Mol. Cell Biol., 20:3224-33, 2000; Appella and Anderson, Signaling to p53: breaking the posttranslational modification code. Pathol. Biol. (Paris), 48:227-45, 2000). The precise mechanisms by which p53 is activated by such multiple regulatory pathways are not completely understood. Generally, however, they are thought to involve post-translational modifications of p53, including ubiquitination, phosphorylation, and acetylation (Giaccia and Kastan, The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev., 12:2973-83, 1998; Appella and Anderson, Signaling to p53: breaking the posttranslational modification code. Pathol. Biol. (Paris), 48:227-45, 2000; Brooks and Gu, Ubiquitination, phosphorylation and acetylation: the molecular basis for p53 regulation. Curr. Opin. Cell Biol., 15:164-71, 2003). In response to DNA damage, for example, p53 is phosphorylated at multiple sites; these phosphorylation events promote p53 stabilization by preventing binding with Mdm2, thereby rendering p53 more resistant to Mdm2-mediated degradation (Shieh et al., DNA damage-induced phosphorylation of p53 alleviates inhibition MDM2. Cell, 91:325-34, 1997; Appella and Anderson, Signaling to p53: breaking the posttranslational modification code. Pathol. Biol. (Paris), 48:227-45, 2000).
By serving as a signal for specific cellular-protein degradation, protein ubiquitination (e.g., mono-ubiquitination, polyubiquitination) plays a critical role in the physiological regulation of many cellular processes (Laney and Hochstrasser, Substrate targeting in the ubiquitin system. Cell, 97:427-30, 1999; Kornitzer and Ciechanover, Modes of regulation of ubiquitin-mediated protein degradation. J. Cell. Phys., 182:1-11, 2000; Hershko et al., The ubiquitin system. Nat. Med., 6:1073-81, 2000; Pickart, C. M., Back to the future with ubiquitin. Cell, 116:181-90, 2004). While polyubiquitination serves primarily as a signal for proteasome-dependent degradation, the functional consequences of mono-ubiquitination are often linked with protein trafficking and other degradation-independent processes (Hicke and Dunn, Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu. Rev. Cell Dev. Biol., 19:141-72, 2003). Deubiquitination, which removes the ubiquitin moiety from ubiquitin-modified proteins, is also now recognized as an important regulatory step (Wilkinson, K. D., Signal transduction: aspirin, ubiquitin and cancer. Nature, 424:738-39, 2003; D'Andrea and Pellman, Deubiquitinating enzymes: a new class of biological regulators. Crit. Rev. Biochem. Mol. Biol., 33:337-52, 1998).
The ubiquitination of p53 was first discovered in papilloma-virus-infected cells, where p53 degradation is mediated by the viral E6 protein (Scheffner et al., The HPV-16 E6 and E6-AP complex functions as an ubiquitin-protein ligase in the ubiquitination of p53. Cell, 75:495-505, 1993). In normal cells, regulation of p53 degradation is governed primarily by Mdm2, an oncoprotein that physically interacts with the N-terminus of p53, and thereby counteracts p53's tumor-suppressor activity (Haupt et al., Mdm2 promotes the rapid degradation of p53. Nature, 387:296-99, 1997; Kubbutat et al., Regulation of p53 stability by Mdm2. Nature, 387:299-303, 1997; Honda et al., Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett., 420:25-27, 1997). By acting as a p53-specific E3 ligase, Mdm2 is critical for degradation of p53, and also induces nuclear export of p53 by mono-ubiquitinating p53 (Freedman et al., Functions of the MDM2 oncoprotein. Cell Mol. Life Sci., 55:96-107, 1999).
Interestingly, transcription of the Mdm2 gene is activated by p53; thus, there is an auto-regulatory loop in which increased Mdm2 production limits p53 induction in response to a variety of cell stresses (Ashcroft and Vousden, Regulation of p53 stability. Oncogene, 18:7637-43, 1999). The critical role of Mdm2 in p53 regulation is best illustrated by studies carried out in mice, wherein inactivation of p53 was shown to rescue completely the embryonic lethality caused by loss of Mdm2 function (Jones et al., Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature, 378:206-08, 1995; Montes de Oca Luna et al., Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature, 378:203-06, 1995).
The stabilization of p53 that occurs in response to oncogene signaling is thought to result from induction of p14ARF, a tumor-suppressor protein that can form a complex with Mdm2, thereby stabilizing both p53 and Mdm2 in vivo (Lowe and Sherr, Tumor suppression by Ink4a-Arf: progress and puzzles. Curr. Opin. Genet. Dev., 13:77-83, 2003). The importance of p14ARF in the p53 pathway is underscored by increased tumor susceptibility of p14ARF-deficient mice (Kamijo et al., Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell, 91:649-59, 1997; Sherr, C.J., The INK4a/ARF network in tumour suppression. Nat. Rev. Mol. Cell Biol., 2:731-37, 2001). MdmX, a member of the Mdm2 family, has recently emerged as another key regulator of p53 function. In contrast to Mdm2, MdmX alone cannot ubiquitinate p53; rather, it stabilizes both p53 and Mdm2 (Stad et al., Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms. EMBO Rep., 2:1029-34, 2001). On this basis, it has been proposed that MdmX and Mdm2 have opposite effects on p53 stability. Mdmx-null mice are embryonic lethal, and the lethality can be rescued in a p53-null background—in a manner reminiscent of Mdm2-deficient mice (Parant et al., Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a non-overlapping pathway with MDM2 to regulate p53. Nat. Genet., 29:92-95, 2001; Finch et al., Mdmx is a negative regulator of p53 activity in vivo. Cancer Res., 62:3221-225, 2002; Migliorini et al., Mdm4 (Mdmx) regulates p53-induced growth arrest and neuronal cell death during early embryonic mouse development. Mol. Cell Biol., 22:5527-38, 2002). Although the precise function of MdmX needs further elucidation (de Graaf et al., Hdmx protein stability is regulated by the ubiquitin ligase activity of Mdm2. J. Biol. Chem., 278:38315-324, 2003; Kawai et al., DNA damage-induced MDMX degradation is mediated by MDM2. J. Biol. Chem., 278:45946-953, 2003; Pan and Chen, MDM2 promotes ubiquitination and degradation of MDMX. Mol. Cell Biol., 23:5113-21, 2003), these studies suggest a critical role for MdmX in the p53 pathway.
As indicated above, evidence suggests that, in cells that retain wild-type p53, other defects in the p53 pathway may play an important role in tumorigenesis. To date, at least one method of treating cancer, by targeting the p53 pathway, has been developed. This treatment involves the stabilization of p53 by inhibiting Mdm2-mediated deubiquitination. It is estimated that 15-30% of all tumor cases exhibit overexpression of Mdm2. However, this enzyme is notoriously difficult to inhibit. Recent studies also imply the existence of an alternative mechanism for p53 stabilization that may function even when the Mdm2-mediated ubiquitination pathway is intact (Ashcroft et al., Regulation of p53 function and stability by phosphorylation. Mol. Cell Biol., 19:1751-58, 1999; Blattner et al., DNA damage induced p53 stabilization: no indication for an involvement of p53 phosphorylation. Oncogene, 18:1723-32, 1999; Dumaz and Meek, Serine15 phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM2. EMBO J., 18:7002-10, 1999; Ashcroft et al., Stress signals utilize multiple pathways to stabilize p53. Mol. Cell Biol., 20:3224-33, 2000).
On the basis of the foregoing, regulation of the p53 pathway is of intense interest, and presents a potential means of diagnosing and treating cancers. Nevertheless, a greater understanding of this pathway, and the regulation of p53 ubiquitination and deubiquitination, would provide a valuable basis upon which new diagnostic and therapeutic methods could be developed.